CN111850004A - Cellulosomal dockerin mutant 36740 with improved activity and application thereof - Google Patents

Cellulosomal dockerin mutant 36740 with improved activity and application thereof Download PDF

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CN111850004A
CN111850004A CN202010730572.0A CN202010730572A CN111850004A CN 111850004 A CN111850004 A CN 111850004A CN 202010730572 A CN202010730572 A CN 202010730572A CN 111850004 A CN111850004 A CN 111850004A
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范翰
薛乐
田文卓
汪俊卿
王瑞明
李楠
姜彦君
王子睿
王大涛
杨翠平
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Qilu University of Technology
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Abstract

The invention relates to a fibrosome docking protein mutant 36740 with improved activity and application, wherein a fibrosome docking protein mutant 36740 is a fibrosome docking protein DocA, the nucleotide sequence is shown as SEQ ID No.3, and the mutant is subjected to site-directed mutagenesis to obtain a docking protein mutant 36740 with improved activity, and the nucleotide sequence is shown as SEQ ID No. 1; the docking protein can be combined and assembled with the mucin more efficiently, and the method has wide application prospect in the construction process of an extracellular multienzyme system of microorganisms.

Description

Cellulosomal dockerin mutant 36740 with improved activity and application thereof
Technical Field
The invention belongs to the field of genetic engineering and enzyme engineering, and particularly relates to a fibrosome dockerin mutant 36740 with improved activity and application thereof.
Background
The cellulosome is a cellulase system existing in anaerobic organisms, is a multienzyme complex structure formed by multiple cellulases and hemicellulases by virtue of an anchoring-adhesion mechanism, is attached to the bacterial cell wall through cell adhesion protein, and has a molecular weight of 2.0x106～2.5x106Da, the natural cellulose material can be efficiently and thoroughly degraded. The fibroid consists essentially of two parts: dockerin (Doc) containing enzymes or other accessory proteins and fibronectin (cohesin, Coh) containing structural proteins. A fibrosome is a cell surface organelle containing a variety of enzymes and associated cofactors which are linked to the enzymes by specific folding subunits. The enzyme molecule is integrated in the cellulosome [ Lytle B L, VolkmanB F, Westler W M, et al. Secondary genomic DNA and calcium-induced folding of the Clostridium thermocellum Domain purified by NMR spectroscopy. Arch Biochem Biophys,2000,379(2): 237-244]Thereby forming a super separationA multifunctional module of the substructure. The fiber-binding domain of the scaffold protein facilitates the binding of the enzyme to the substrate [ Yague E, Beguen P, Aubert J P. nucleotide sequence and deletion analysis of the cellulose-encoding gene CelH of cloning thermal cell. Gene,1990,89: 61-67]. The fibrosomes can also form a poly-fibrosome through mutual anchoring effect, and a layer of glycoprotein coat is wrapped to form a protruding structure on the surface of the cell.
Cellulosome tissues various degrading enzymes form multienzyme complexes, thereby efficiently degrading cellulosic materials. The mode of action of this supramolecular structure is key to understanding cellulose degradation and utilization of cellulose resources. Interactions between proteins in the fibrosomes, morphogenesis of the organization structure of the fibrosomes, etc. Researches find that the cellulosome can greatly improve the degradation rate of cellulose (which is several times higher than that of the cellulose degraded by pure cellulase), so that the utilization rate of the cellulose is obviously improved. However, the difference of the cellulosome produced by different cellulose-degrading bacteria is large, mainly due to the difference of the self-assembly modes, the difference of the structures [ luohui, enemy Tianlei, Lei, etc.. the research progress of cellulose anaerobic degradation [ J ]. Chinese biogas, 2008, 26 (2): 3-9].
Cellulosomes are usually present on the cell surface and are degraded by sufficient contact of the cellulose binding units with the cellulose surface. Cellulose is a renewable resource which is found at present in the largest quantity and has high utilization value, but only a small part of cellulose is developed and utilized by human beings, and most of cellulose becomes waste [ wu shui picture, penhui, shore blue ] research progress of crystalline cellulose degrading enzyme [ J ] Anhui agricultural science, 2007, 35 (9): 2532-2534]. The cellulose is extremely difficult to dissolve in water and degrade, and due to the existence of cellulose bodies, the cellulose can be efficiently degraded, so that the cellulose is converted into chemical products and fuels, and the environment and the ecological green sustainable development are realized. Although various cellosome elements are found at present, research and development of a more efficient extracellular self-assembly system and a manufacturing method thereof are still crucial to further application of the cellosome and the elements thereof.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a mutant 36740 of a cellulosome dockerin with improved activity and application thereof.
The technical scheme of the invention
A fibrosome dockerin mutant 36740 is a fibrosome dockerin DocA, the nucleotide sequence is shown in SEQ ID NO.3, and site-directed mutagenesis is performed to obtain a dockerin mutant 36740 with improved activity, and the nucleotide sequence is shown in SEQ ID NO. 1.
A fibrosome docking protein mutant 36740 is a fibrosome docking protein DocA, the amino acid sequence is shown in SEQ ID NO.4, and the mutant carries out site-directed mutagenesis to obtain a docking protein mutant 36740 with improved activity, and the amino acid sequence is shown in SEQ ID NO. 2.
The DocA is derived from Clostridium thermocellum (Clostridia thermocellum) (GenBank:2CCL _ B), the nucleotide sequence of the DocA is shown as SEQ ID NO.3, and the amino acid sequence of the DocA is shown as SEQ ID NO. 4.
D in the above amino acid sequence40VDKNGSINAAD51Mutation to D40TSNDGYINAAD51。
The preparation method of the fibrosome dockerin mutant 36740 comprises the following steps:
carrying out site-directed mutagenesis by taking a nucleotide sequence in the docking protein as shown in SEQ ID No.3 to obtain a docking protein mutant 36740, designing a site-directed mutagenesis primer on the basis of the nucleotide sequence as shown in SEQ ID No.1, carrying out PCR by taking a pET28a (+) vector carrying a cellosome docking protein gene as a template to construct a recombinant mutant plasmid, transforming the mutant plasmid into escherichia coli BL21(DE3), selecting a positive clone for fermentation, collecting thalli after the fermentation is finished, crushing the thalli, and purifying to obtain the cellosome docking protein mutant.
According to the invention, in the preferable preparation method, the dockerin is mutated into a dockerin mutant 36740 by the dockerin with an amino acid sequence shown as SEQ ID No.4, and the amino acid sequence is shown as SEQ ID No. 2.
Preferably, in the above preparation method, after the fermentation is completed, the bacterial cells are collected by centrifugation, disrupted by sonication, and purified by affinity chromatography to obtain the mutant of the fibrosome dockerin.
According to a preferred embodiment of the present invention, the nucleotide sequence of the PCR amplification primer of dockerin mutant 36740 is as follows:
36740-F：GTGCCGAT accagcaatgat GGC tat ATTAATGCC SEQ ID NO.7；
36740-R：GGCATTAAT ata GCC atcattgctggt ATCGGCACGGGCTTT SEQ ID NO.8。
the lower case letters in the primer nucleotide sequence are mutation sites.
Further preferably, in the preparation method, the PCR reaction system:
1 μ L of plasmid vector template, 2 μ L of forward mutation primer F, 2 μ L of reverse mutation primer R, 10 μ L of 5 XFastmutation buffer, 1 μ L of Fastmutation DNA Polymerase, ddH2O34μL。
Further preferably, in the preparation method, the PCR reaction conditions are as follows:
pre-denaturation at 95 ℃ for 2 min; 94 ℃ for 20sec, 55 ℃ for 10sec, 68 ℃ for 3min, 18 cycles; extension was supplemented at 68 ℃ for 5 min.
According to the invention, preferably, after the PCR reaction is completed, the original template in the PCR amplification product needs to be digested by Dnp I enzyme, and after the digestion reaction system is mixed uniformly, the original template is digested for 1 hour at 37 ℃ to obtain the vector to be transformed.
Further preferably, the digestion system:
PCR amplification product 40. mu.L, Dnp I enzyme 1. mu.L.
Further preferably, the transformation of the mutant vector comprises transforming the above-mentioned vector to be transformed into E.coli BL21(DE3) cells, and plating the transformant with a medium containing kanamycin (50. mu.g.mL)-1) After overnight culture at 37 ℃, single colony is picked out, sequencing and verification are carried out, and positive mutant is screened to obtain recombinant escherichia coli 36740-BL 21.
More preferably, the mutant is expressed and purified by inoculating the strain containing kanamycin (50. mu.g.mL) with the strain whose accuracy was verified as described above-1) In the liquid LB medium of (1), 3Culturing at 7 deg.C overnight, transferring to liquid LB culture medium, culturing at 37 deg.C to OD600When the concentration is approximately equal to 1, 1 mu m.mL is added-1The IPTG was induced at 26 ℃ for 8 hours at 200rpm, the induced cells were collected, then the cells were resuspended in 2 XPBS buffer, disrupted by ultrasonication, centrifuged at 10000rpm for 10 minutes, the protein in the supernatant after centrifugation was purified by a nickel ion affinity column and desalted by dialysis with PBS-EP + buffer, to obtain purified mutant fibrinosomal dockerin 36740.
Use of the aforementioned mutant of cellulosome dockerin 36740 for interacting with fibronectin to construct a protein complex.
Preferably, according to the invention, the concentration of calcium ions required in the above-mentioned applications is 10-4M～10-2M。
The invention has the beneficial technical effects
DocA is a fibrosome docking protein currently reported to have high docking activity and has been widely used. The invention provides a dockerin mutant 36740 with calcium ion concentration of 5 × 10-4Docking activity with mucin CohA at M was 3.68 times that of DocA and was found at a calcium ion concentration of 10-4M～10-2M shows higher activity in the environment, and the better activity can improve the assembly efficiency and the finishing strength of the cellulosome, thereby having wider application prospect.
Drawings
FIG. 1 is a line graph showing the binding capacity of the mutant fibronectin and fibronectin at different calcium ion concentrations in example 4
Detailed Description
The invention is further illustrated with reference to specific examples, without limiting the scope of protection.
Sources of materials
The vectors DocA-pET28a and CohA-pET28a were extracted from Escherichia coli DH 5. alpha. or stored in the Escherichia coli DH 5. alpha. in the laboratory, which is an important laboratory in microbial engineering, Shandong, university of Qilu Industrial science, and those skilled in the art can construct the vectors according to the prior art or purchase them from the laboratory.
The contents of the examples, which are not specified in specific conditions, were carried out under conventional conditions; the reagents or instruments used are not indicated by the manufacturer, and are all common commercial products.
Example 1
Mutant primer design and mutant vector construction
Primer design of dockerin mutants was performed using dockerin DocA (vector DocA-pET28a, nucleotide sequence shown in SEQ ID NO. 9) ligated to pET28a (+) vector, respectively, as a template (Table one). Wherein the DocA is derived from Clostridium thermocellum (Clostridia thermocellum) (GenBank:2CCL _ B), and the optimization of nucleotide sequence is carried out according to the codon preference of Escherichia coli, the DocA nucleotide sequence is shown as SEQ ID NO.3, and the DocA amino acid sequence is shown as SEQ ID NO. 4.
TABLE-mutant primer Table
Figure BDA0002603104980000041
Note: lower case letters are mutation sites.
And accurately adding the plasmid DocA-pET28a, the designed mutation primer, the PCR enzyme and the buffer according to the addition amount of each component in the second table to prepare a PCR reaction solution, and carrying out PCR amplification according to the PCR reaction conditions in the third table.
PCR reaction system for epidiaschisis
Figure BDA0002603104980000042
PCR reaction conditions for the epitrimutation
Figure BDA0002603104980000043
Figure BDA0002603104980000051
After the PCR reaction is completed, the original plasmid template methylated by Escherichia coli DH5 alpha in the PCR product needs to be digested by Dnp I enzyme, the digestion reaction system is shown in Table four, and after the system is prepared and fully mixed, the mixture is digested for 1h at 37 ℃.
TABLE IV digestion System
Figure BDA0002603104980000052
Taking escherichia coli BL21(DE3) competence at-80 ℃ and quickly dissolving the enzyme in ice at 4 ℃, sucking a proper amount of enzyme digestion solution by a micropipette and injecting the enzyme digestion solution into the molten escherichia coli BL21(DE3) competence, uniformly mixing the tube walls of the flickers, accurately heating the mixture for 90sec at 42 ℃, carrying out ice bath for 2min, adding 400 mu L of liquid LB culture medium (1% peptone, 1% yeast extract powder, 0.5% NaCl and the balance of water) into an ultraclean workbench after the ice bath, recovering the mixture in a shaking table at 37 ℃ for about 1h, centrifuging the mixture for 5min at 4000rpm after the recovery is finished, removing 400 mu L of supernatant, and directly coating the rest 200 mu L of the mixture to a solution containing kanamycin (50 mu g. mL)-1) After overnight culture on the solid medium (1% peptone, 1% yeast extract, 0.5% NaCl, 2% agar, balance water), a single colony was picked up and cultured, and sent to sequencing company for sequencing and verifying the amino acid sequence D in DocA40VDKNGSINAAD51Mutation to D40TSNDGYINAAD51(ii) a Screening out positive clones to obtain recombinant Escherichia coli 36740-BL 21.
Example 2
Inducible expression and purification of muteins
The correctly verified strain was inoculated to a strain containing kanamycin (50. mu.g.mL)-1) The cultured cells were cultured overnight at 37 ℃ in 50mL of liquid LB medium, and then transferred to a new 50mL of liquid LB medium at 37 ℃ in an amount of 2% by volume, and cultured to OD600When the concentration is approximately equal to 1, the mixture is added until the final concentration is 1 mu m.mL-1The IPTG was placed in a shaker at 26 ℃ and 200rpm for 8 hours, and the induced cells were collected. The induced cells were resuspended in 10mL 2 XPBS buffer, disrupted by ultrasonication, centrifuged at 10000rpm for 10min, and the protein in the supernatant after centrifugation was purified by a nickel ion affinity column and desalted by dialysis using PBS-EP + buffer (from GE). Purified fibrosome dockerin 36740 was obtained.
Example 3
Construction and induced expression of expression vector of fibronectin
Transformation of the CohA-pET28a vector was carried out in the same manner as in example 1 using pET28a (+) vector to which the cellulosome fibronectin CohA was ligated, and similarly transformation of DocA-pET28a vector was prepared to obtain recombinant E.coli DocA-BL21 and CohA-BL21, and inducible expression and purification of genes were carried out in the same manner as in example 2. Purified fibrosome mucin CohA, fibrosome dockerin DocA was obtained. Wherein the cellulosome mucin CohA is derived from a Clostridium thermocellum (Clostridium thermocellum) scaf gene (GenBank: MH049738.1), the nucleotide sequence of the CohA is shown as SEQ ID NO.5, and the amino acid sequence of the CohA is shown as SEQ ID NO. 6.
Example 4
Analysis of binding Capacity of Fibrosomal docking protein and mucin at different calcium ion concentrations
Figure BDA0002603104980000061
Analyzing the binding capacity between the mutant of the cellosome docking protein and the fibronectin with different calcium ion concentrations by using a biomacromolecule interaction instrument, selecting a proper CM5 chip as an anchoring chip, calculating the approximately required concentration of the protein CohA according to a formula, performing gradient dilution on acetic acid-sodium acetate buffer solutions with different pH values according to the approximately protein concentration to serve as the protein to be anchored, determining the optimal anchoring concentration and pH value according to the anchoring condition, and then anchoring the CohA according to the mucoprotein CohA in Biacore micromolecule application operation manual. The anchored chip was loaded into a molecular interaction apparatus in 1 XPBS-HP + solution (available from GE), and the dockerin and mutant were diluted to nearly the same anchoring concentration as the protein CohA and combined with different concentrations of CaCl2After standing at 4 ℃ for 30min, the binding was determined on the machine and judged according to the AbsResp value (i.e., the binding interaction intensity value), and the results are shown in fig. 1 (table v). The results indicate that dockerin mutant 36740 had calcium ion concentrations greater than or equal to 10, relative to unmutated dockerin DocA-4Binding ability to mucin CohA at MShows enhanced activity at a calcium ion concentration of 5X 10-4The docking activity of the binding protein CohA at M was 3.68 times that of DocA.
Analysis of binding Capacity of Fivessilebody dockerin mutant and mucin at different calcium ion concentrations
Figure BDA0002603104980000062
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gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160
tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220
agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280
tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340
caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400
ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520
gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580
gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640
aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700
ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760
acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820
ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880
tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940
tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000
cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060
gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120
ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180
catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240
ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300
gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360
gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420
ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480
atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat3600
tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660
ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720
aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780
atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840
cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900
gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960
tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020
agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080
gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140
ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200
catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260
tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320
tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380
gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440
ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500
tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560
catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620
cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680
tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740
ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800
ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860
cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920
gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980
aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040
ttttgtttaa ctttaagaag gagatatacc atggtgctgc tgggtgacgt gaatggtgac 5100
ggtaccatta atagcaccga tctgaccatg ctgaaacgtt ctgttctgcg tgccattacc 5160
ctgaccgatg atgccaaagc ccgtgccgat gtggataaaa atggcagcat taatgccgcc 5220
gatgttctgc tgctgtctcg ctatctgctg cgtgttattg ataaaggagg aggcggctcg 5280
ggaggaggcg gctcgggagg aggcggctcg catcatcatc atcatcatta agaattcgag 5340
ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc 5400
tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc 5460
ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat 5520
atccggat 5528

Claims (10)

1. A fibrosome dockerin mutant 36740 is characterized in that a fibrosome dockerin DocA with a nucleotide sequence shown in SEQ ID NO.3 is subjected to site-directed mutagenesis to obtain a dockerin mutant 36740 with improved activity, wherein the nucleotide sequence is shown in SEQ ID NO. 1.
2. A fibrosome docking protein mutant 36740 is a fibrosome docking protein DocA, the amino acid sequence is shown in SEQ ID NO.4, and the mutant carries out site-directed mutagenesis to obtain a docking protein mutant 36740 with improved activity, and the amino acid sequence is shown in SEQ ID NO. 2.
3. A method of making the dockerin mutant 36740 of any one of claims 1-2, comprising the steps of:
carrying out site-directed mutagenesis by taking a nucleotide sequence in the docking protein as shown in SEQ ID No.3 to obtain a docking protein mutant 36740, designing a site-directed mutagenesis primer on the basis of the nucleotide sequence as shown in SEQ ID No.1, carrying out PCR by taking a pET28a (+) vector carrying a cellosome docking protein gene as a template to construct a recombinant mutant plasmid, transforming the mutant plasmid into escherichia coli BL21(DE3), selecting a positive clone for fermentation, collecting thalli after the fermentation is finished, crushing the thalli, and purifying to obtain the cellosome docking protein mutant.
4. The method of claim 3, wherein the dockerin is mutated to dockerin mutant 36740 from dockerin having the amino acid sequence shown in SEQ ID No.4 and the amino acid sequence shown in SEQ ID No. 2.
5. The method according to claim 3, wherein the strain is collected by centrifugation after the fermentation is completed, and the strain is subjected to ultrasonication and affinity chromatography for purification to obtain the mutant of the fibrosome dockerin.
6. The method of claim 3, wherein the nucleotide sequence of the PCR amplification primer of dockerin mutant 36740 is as follows:
36740-F is shown in SEQ ID NO. 7;
36740-R is shown in SEQ ID NO. 8.
7. The method according to claim 6, wherein the PCR reaction system comprises:
1 μ L of plasmid vector template, 2 μ L of forward mutation primer F, 2 μ L of reverse mutation primer R, 10 μ L of 5 XFastmutation buffer, 1 μ L of Fastmutation DNA Polymerase, ddH2O34μL。
8. The method of claim 7, wherein the PCR reaction conditions are:
pre-denaturation at 95 ℃ for 2 min; 94 ℃ for 20sec, 55 ℃ for 10sec, 68 ℃ for 3min, 18 cycles; supplementary extension at 68 ℃ for 5 min;
preferably, after the PCR reaction is finished, the original template in the PCR amplification product needs to be digested by Dnp I enzyme, and after a digestion reaction system is uniformly mixed, the original template is digested for 1 hour at the temperature of 37 ℃ to obtain a vector to be transformed;
preferably, the digestion system:
PCR amplification product 40. mu.L, Dnp I enzyme 1. mu.L;
preferably, the transformation of the mutant vector is carried out by transforming the above-mentioned vector to be transformed into E.coli BL21(DE3) cells, plating the transformant with a transformant containing kanamycin (50. mu.g.mL)-1) After overnight culture at 37 ℃ on the LB solid medium, selecting a single colony, carrying out sequencing verification, screening a positive mutant, and obtaining a recombinant large intestine rodBacterium 36740-BL 21;
preferably, the mutant is expressed and purified by inoculating the correctly verified strain containing kanamycin (50. mu.g.mL)-1) The cultured cells were cultured overnight at 37 ℃ in the liquid LB medium, and then transferred to the liquid LB medium and cultured to OD at 37 ℃600When the concentration is approximately equal to 1, 1 mu m.mL is added-1The IPTG was induced at 26 ℃ for 8 hours at 200rpm, the induced cells were collected, then the cells were resuspended in 2 XPBS buffer, disrupted by ultrasonication, centrifuged at 10000rpm for 10 minutes, the protein in the supernatant after centrifugation was purified by a nickel ion affinity column and desalted by dialysis with PBS-EP + buffer, to obtain purified mutant fibrinosomal dockerin 36740.
9. Use of the dockerin mutant 36740 of any one of claims 1-2 to interact with mucin to construct a protein complex.
10. The use of claim 9, wherein the desired calcium ion concentration is 10-4M～10-2M。
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